1. Field of the Invention
The present invention relates to the identification and purification of herpes proteases and to nucleic acid segments coding for such proteases. The present invention also relates to methods of selecting candidate substances that are able to inhibit the function of these proteases and the use of such inhibitors to detect and treat viral infections.
2. Description of Related Art
a. Viral Infections Pose Major Health Problems
Treatment and prevention of viral infections is a major medical goal. To understand the state of the art in developing methods of treating and preventing viral infections, it is important to understand the structure and function of infectious virus. A virus is a small genetic element that contains either single or double-stranded DNA or RNA and can alternate between two distinct states: intracellular and extracellular. A virus is a obligatory intra-cellular parasite that cannot reproduce by itself. In effect, the virus takes over the biosynthetic machinery of the host cell and uses it for viral synthesis. Some of the protein products of the viral DNA are special enzymes or inhibitory factors that stop host cell metabolism, but most viral-encoded proteins are used in the construction of new virions. Protein synthesis is directed by the virus to produce necessary components for its replication and packaging, e.g. the capsid. These components must be assembled in an order depending on the virus, and new particles must escape from the cell if they are to infect other cells.
The general steps of the intracellular viral replication (lytic cycle) are:
1. attachment of the virus to a host cell (absorption);
2. penetration of the virus or its nucleic acid into the host cell;
3. replication of the viral nucleic acid;
4. production of viral proteins and other essential components;
5. assembly of viral nucleic acid and protein components; and
6. release of mature virion particles from the host cell.
The overall result of the lytic cycle is new virus particles and dead host cells because the virus has appropriated the vital forces of the host. In certain types of infection, such as that caused by herpes, there may be a latent period wherein the virus resides in the host cell.
Elucidation of viral genetic systems opens the door to investigations on the mechanisms of viral infection and replication which are not simply of academic interest, but are directed toward detection, prevention, and treatment of viral caused diseases. Host resistance to viral infection may occur through absence of a viral-receptor site to prevent attachment of the virus to the host cell; destruction of the viral nucleic acids after they are injected into the host cell, for example, by cleavage of viral nucleic acids by host enzymes; inhibition of essential viral protein synthesis; or destruction of viral proteins after their formation in the host cell.
Development of antiviral drugs to supplement natural resistance is a major commercial objective whose goal is to counteract the devastating effects of many viral infections on humans. Unfortunately, treatments available to date are inadequate for most types of virus. For example, interferons are cellular antiviral substances, low molecular weight proteins, that prevent viral multiplication. However, interferons tend to be host specific, not viral specific, and have no effect on host cells already infected. Also, they can be toxic at high concentrations.
The target of antiviral drugs may be enzymes uniquely specified by the virus. For example, a major target for attack on HIV infections which cause AIDS, is the enzyme reverse transcriptase. Inhibiting this enzyme effects blockage of viral replication. Unfortunately, resistance develops to these drugs, e.g. to AZT, and is a major limitation of such treatment. The anti-herpes simplex virus drugs currently on the market are directed against enzymes which synthesize viral DNA (e.g. acyclovir). Because of emergence of resistance to these drugs, there is considerable interest in new targets.
Production of viral proteins is of particular interest as a stage where the virus may be attacked. In the extracellular or infectious state, the basic structure of viruses consists of a nucleic acid core surrounded by proteins (nucleocapsid). Some viruses also have an envelope that is external to the nucleocapsid, and contains lipids and protein. The protein coat is called the capsid. Many different proteins may constitute the capsid, depending on the virus. Some viruses encode 3-10 proteins, others more than 200. Virus particles are called virions; their role is to protect the viral nucleic acid when transferred from the cell in which it replicated to a new host cell. After transfer to the host cells, the viral intracellular state begins, and replication of the virus is potentiated. A number of non-herpes viruses appear to express proteases with cleavage site specificity which are potential targets for therapeutic intervention. However, no such protease has previously been identified for the herpes virus, a particularly widespread infectious agent for which treatment and prevention methods are grossly inadequate.
b. The Herpes Family
The family of herpes virus includes animal viruses of great clinical interest because they are the causative agents of many diseases. Epstein-Barr virus has been implicated in cancer initiation; cytomegalovirus is the greatest infectious threat to AIDS patients; and Varicella Zoster Virus, is of great concern in certain parts of the world where chicken pox and shingles are serious health problems. A worldwide increase in the incidence of sexually transmitted herpes simplex (HSV) infection has occurred in the past decade, accompanied by an increase in neonatal herpes. Contact with active ulcerative lesions or asymptomatically excreting patients can result in transmission of the infective agent. Transmission is by exposure to virus at mucosal surfaces and abraded skin, which permit the entry of virus and the initiation of viral replication in cells of the epidermis and dermis. In addition to clinically apparent lesions, latent infections may persist, in particular in nerve cells. Various stimuli may cause reactivation of the HSV infection. Consequently, this is a difficult infection to eradicate. This scourge has largely gone unchecked due to the inadequacies of treatment modalities.
c. Herpes Simplex Virus (HSV)
Herpes simplex viruses subtypes 1 and 2 (HSV-1, HSV-2), are herpes viruses that are among the most common infectious agents encountered by humans (Corey and Spear, 1986; Whitley, 1990). These viruses cause a broad spectrum of diseases which range from relatively insignificant and nuisance infections such as recurrent herpes simplex labialis, to severe and life-threatening diseases such as herpes simplex encephalitis (HSE) of older children and adults, or the disseminated infections of neonates. Clinical outcome of herpes infections is dependent upon early diagnosis and prompt initiation of antiviral therapy. However, despite some successful therapy, dermal and epidermal lesions recur, and HSV infections of neonates and infections of the brain are associated with high morbidity and mortality. Earlier diagnosis than is currently possible would improve therapeutic success. In addition, improved treatments are desperately needed.
Extrinsic assistance has been provided to infected cells, in particular, in the form of chemicals. For example, chemical inhibition of herpes viral replication has been effected by a variety of nucleoside analogues such as 5-fluorodeoxyuridine (FUDR), 5-iododeoxyuridine, 5-iododeoxyuridine, thymine arabinoside, and the like. Some protection has been provided in experimental animal models by polyspecific or monospecific anti-HSV antibodies, HSV-primed lymphocytes, and cloned T cells to specific viral antigens (Corey and Spear, 1986). However, no satisfactory treatment has been found.
Proteases have not been identified in the herpes viruses, but there is some knowledge of the biology of the herpes virus family. Herpes viruses are double stranded DNA viruses that replicate in host cell nuclei. The herpes virion is constituted from over 30 different proteins which are assembled within the host cell. About 6-8 are used in the capsid. The preferred host cells for herpes viruses are vertebrate cells.
The herpes simplex virus 1 (HSV-1) the genome specifies an abundant capsid protein complex which in denaturing gels forms multiple bands due to different molecular weights of the component proteins. Some preliminary identification of these proteins has been reported. A set of herpes simplex virus 1 (HSV-1) capsid proteins was reported by Gibson and Roizman (1972, 1974). A genetically and immunologically related family of viral capsid proteins identified by their migration bands in denaturing gels has been designated infected-cell proteins 35(ICP35). (Braun et al., 1983, 1984) At least four major and a number of minor bands in one-dimensional denaturing polyacrylamide gels, and numerous spots in two-dimensional gels, have been reported.
Braun et al. (1984) using a panel of monoclonal antibodies exemplified by H745 reported that ICP35 proteins are processed post-translationally into at least 6 species (ICP35a,b,c,d,e,f) differing in electrophoretic mobility on SDS polyacrylamide gels. Although characterized by different molecular weights, this group of virus polypeptides are detected by the same monoclonal antibodies and are coded by a region in the HSV-1 genome. Empty capsids do not contain these polypeptides. A set of proteins possibly analogous to ICP35 was reported by Preston et al. (1983).
Nucleotide sequencing has been performed on the HSV-1 genome, and attempts, generally unsuccessful, have been made to correlate various capsid proteins to sequences of the genome. It has been proposed that the ICP35 proteins are encoded by the open reading frame designated UL26 (McGeoch, et al., 1988). In the present invention it is shown that this prediction was incomplete and not correct. Crude mapping of the region encoding ICP35 was attempted by Braun et al. (1984) on the basis of the analysis of HSV-1xc3x97HSV-2 intertypic recombinants. These authors proposed that ICP35 is encoded by a region located between the genes specifying thymidine kinase (UL23) and glycoprotein B (UL27). This is not a very specific prediction because it covers an area including four genes.
The present invention resulted from a successful search for a new virus target for therapy. The search began by choice of the HSV-1 ICP35 protein family as a substrate. A protease target for antiviral chemotherapy, in particular, as applied to herpes virus infections, was identified in this fashion. Methods of preparing and detecting the protease, methods for selecting inhibitors of the protease, as well as detection and treatment protocols based on inhibiting the protease, are also aspects of the present invention.
This invention relates to the identification, purification and manipulation of viral proteases to effect treatment and prevention of viral infections. The proteases of the present invention are further defined as serine proteases with the properties expected of this category of protease. A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds in which there is an essential serine residue at the active site. (White, Handler and Smith, 1973). Differences among serine proteases include an arrangement of a triad of catalytic residues, e.g., in both trypsin and subtilisin serine proteases, Asp, His, and Ser are the amino acids of the catalytic triad. However, in trypsin-like serine proteases, they are arranged His, Asp, Ser, whereas subtilisin-like proteases are arranged Asp, His, Ser. There are also differences in the relative spacing of these key residues. In addition, there are other evolutionarily conserved features of these proteases which allow them to be identified as serine proteases and subsequently classified. The presence of the catalytically important Asp, His, and Ser residues are the crucial tests, however, for membership and classification in the serine proteases.
The proteases of the present invention appear to be essential for development of the capsid of the virus. Consequently, inhibiting the protease action will lead to disruption of the lytic cycle of the virus. Obviously, such proteases are optimal targets for antiviral therapy. In particular, the target is useful for attacks on the herpes virus for which no protease has heretofore been reported. The present invention relates more particularly to the identification, purification, and manipulation of herpes serine proteases, and to the use of inhibitors of the proteases to detect and treat herpes infections.
In an illustrative embodiment, a protease has been purified from HSV-1, a subtype of the herpes simplex virus. The apparent molecular weight of this protease as determined by SDS-polyacrylamide PAGE gel electrophoresis is approximately 75-85 kd, generally about 80 kd. The herpes protease is further characterized as having an amino acid sequence of approximately 450-635 amino acids. However, these ranges are flexible. For example, the 635 amino acid sequence SEQ ID NO: 2 may have at least 125 amino acids removed from its carboxyl end and still maintain its serine protease activity. In the 635 amino acid embodiment SEQ ID NO: 2 the protease cleavage site is located at a position about 18-25 amino acids from the carboxyl terminus, preferably about 20 amino acid from the carboxyl terminus. The proteases are obtained either from cells injected with HSV-1 and 2, cells transfected with a DNA sequence encoding the protease, or in purified form by being synthesized in vitro in cell free systems, using a reticulocyte lysate, for example, from a rabbit. The latter is a xe2x80x9cribosome machinexe2x80x9d that permits production of a purified protein encoded by the nucleic acid sequence added to the xe2x80x9cmachine.xe2x80x9d After synthesis of the protein, the protein will migrate as a single band on a denaturing gel and is readily detected with 35S labelled methionine. After about 5 hours of protein synthesis, two bands will result. As demonstrated in subsequent sections, the second band is a self-cleavage product of the first.
The present invention also relates to nucleic acid segments which are capable of coding for the herpes proteases described herein. In an illustrative embodiment, extensive manipulation of nucleic acid sequences within the HSV-1 genome has revealed the secrets of viral mechanisms and allowed isolation and purification of useful nucleic acid segments and their expression products. Examples of such manipulation include incorporation of selected segments of the nucleic acid sequence with appropriate promoters and tracers into plasmids to determine the actions and interactions of the genetic regions, their expression products, and mechanisms of control over their expression. These specially designed plasmids are aspects of the invention.
Another aspect of this invention is the coding domain in the nucleic acid segment for the family of herpes simplex virus 1 capsid proteins designated ICP35. This newly defined coding region has been designated UL26.5. In an illustrative embodiment of the coding sequence for ICP35 proteins, the segment has been demarcated by the restriction endonuclease cleavage sites Hpa-I and Pst-I. These two cleavage sites are located at map positions +832 and +2761. These map locations are defined as distances from the transcription initiation site of the UL26 open reading frame in the HSV-1 genome. This position has been designated +1. The gene coding for the ICP35 proteins also comprises those sequences that are downstream from the KpnI site which is at map position +2104, and continue all the way to a poly A site at position +2138.
The nucleic acid segments in the present invention are further defined as having overlapping open reading frames for the protease and the ICP35 proteins. These overlapping segments are xe2x80x9c3xe2x80x2co-terminal.xe2x80x9d The first segment SEQ ID NO: 1, the longer of the two, codes for a first protein. This first protein has a molecular weight of approximately 75-85 kd. The second open reading frame, the smaller of the two overlapping open reading frame sequences encodes a second protein that has an apparent approximate molecular weight of 40-55 kd as determined by SDS polyacrylamide gel electrophoresis, preferably 45 kd. The first protein is defined to encode a least a proteolytic module which is capable of cleaving an amino acid sequence in accordance with serine protease action. The substrate capable of being cleaved may be either the protease sequence itself that is encoded by the UL26 gene, or the ICP35 precursor proteins, which have previously been designated ICD35 c, d based on migration in one and two dimensional gels.
A nucleic acid segment, either DNA or RNA, coding for the second protein includes approximately 990 base pairs in an HSV-1 embodiment. In certain applications, the segment encodes need include only the sequences which upon cleavage will yield ICP35 e and f. In other applications, only the cleavage site per se may be desired to be encoded, for example in the candidate inhibitor assay described subsequently. In an exemplary embodiment, the nucleic acid segment includes those segments essentially as set forth in FIG. 1A, line 5, of the present specification, or its functional equivalent. A functional equivalent for purposes of this invention may be defined as a segment of nucleic acids which encodes an amino acid sequence that contains a catalytic serine protease coding domain capable of producing a protease which cleaves ICP35. The nucleic acid segment may include a sequence either smaller or larger than those illustrated in FIG. 1, for example, it may correspond to an approximately 14 nucleotide base pair long region which is capable of hybridizing to the nucleic acid segments of FIG. 1 under stringent conditions. Small segments such as this may be used as probes to detect the presence of the protease coding region.
A nucleic acid segment may also be an mRNA sequence for the protease or the ICP35 proteins. The mRNA for the latter is transcribed at approximate nucleotide position +1000 (a designated distance from the herpes UL26 transcription initiation site which is designated +1) to position +2138. An mRNA is translated from the methionine initiation codon which is located at +1099. Smaller or larger segments are also contemplated depending on the use to which the segment is employed. These mRNA segments are useful to synthesize the ICP35 cleavage site.
Various nucleic acid segments within the herpes genome have been isolated and cloned. A nucleic acid segment coding for a herpes protease may be obtained from the herpes genome from a region corresponding to map locations of the UL26 herpes open reading frame SEQ ID NO: 1. A smaller segment contained within the protease coding segment lies between the thymidine kinase gene and glycoprotein gB gene and includes a DNA sequence from position +832 to +2138. This coding sequence not only may be expressed as the ICP35 herpes capsid family proteins, but includes a promoter sequence for regulating the ICP35 coding sequence.
The ICP35 promoter has been isolated and shown to be an extremely active promoter, as is evidenced by the observation that increased numbers of copies of the substrate are produced by the ICP35 encoding unit compared to the production of the protease from the longer UL26 open reading frame. It includes between 135 and 168 base pairs and maps between position +832 and +1000 in the first open reading frame of UL26. This promoter region is capable of initiating expression of the ICP35 proteins. It is also useful in driving expression at an increased rate of other nucleic acid sequences, for example herpes simplex genes US5 and UL10.
Isolation and manipulation of the coding sequence for the ICP35 proteins was an important achievement of the present invention because these proteins are used in construction of the herpes virus capsid. To become functional members of the capsid, the ICP35 protein precursor ICP35 c, d must be cleaved by the herpes protease produced by the UL26 coding sequence, to yield e and f. This protease is capable of effecting cleavage of the precursor proteins even when both genes encoding for the protease and the precursor are in a trans position.
The nucleic acid segments of the present invention may be carried in a recombinant expression vector capable of expressing virus serine proteases which are present in a host cell. Examples of such nucleic acid segments are those shown for the herpes virus, HSV-1 in FIG. 1A designated as A-Z of the present specification. These nucleic acid segments may be operably linked to the non-herpes derived components in plasmids A-Z or their functional equivalents. These components may include promoters such as those capable of controlling the herpes xcex14, the ICP35 genes or their functional equivalent. The recombinant expression vectors may comprise as a promoter either a eukaryotic or prokaryotic promoter, and may include a polyadenylation signal at position 3xe2x80x2 of the carboxyl terminal amino acid. The promoter may be within a transcriptional unit of the encoded protein. Vectors may also include markers which have been used for the analysis presented herein. Examples of host cells are BHK cells, Vero, E. coli or other eukaryotic or prokaryotic cells known to those of skill in the art to permit expression of transferred vectors according to the present invention.
This invention further relates to methods of preparing a herpes protease. An embodiment of such methods includes the following steps:
(1) Preparing a nucleic acid segment which encodes the herpes protease; and
(2) Allowing the segment to be expressed in order to produce the protein.
In an illustrative embodiment the method of preparing a protease includes use of a host cell into which the nucleic acid segment has been transferred. The host cell is cultured under conditions suitable for expression and the protein is thereby expressed. The method may also include a step wherein the protein is isolated and purified by methods well known to those skilled in the art. The degree of purification required will depend on the application for which the protein is intended. Alternatively, the nucleic acid segment may be expressed in a cell free system, such as a rabbit reticulocyte lysate, or synthesized by an automated protein synthesizer as referenced herein.
A nucleic acid segment which codes for the herpes protease or for the ICP35 proteins may be prepared by obtaining viral genomic DNA from cells which are infected with herpes, applying the proteolytic site containing nucleic acid sequence region within the nucleic acid of interest, and preparing recombinant clones which include such amplified nucleic acid sequences. The clones may be then selected to contain the desired amplified nucleic acid segments by employing monoclonal antibodies directed to at least the region coding for the proteolytic domain of the protease or the cleavage site of the ICP35 protein to screen such clones. Other cloning and clone screening techniques well known to those of skill in the art are also suitable (Maniatis).
This invention also relates to a method for cleaving a herpes molecule which includes the steps of treating the molecule with the protease under conditions effective for cleavage. Such conditions are those in which serine proteases generally operate.
In an exemplary embodiment, methods for detecting the herpes protease in tissue samples consist of preparing antibodies directed against the protease, labelling these antibodies, contacting tissue samples with the labelled antibody, and detecting the labelled antibody protein heteroconjugate by standard techniques well known to those of skill in the art. These labels may be fluorescent labels or radioactive labels.
One of the methods for detecting the nucleic acid segments in biological samples is to prepare a nucleotide probe that is capable of hybridizing to a nucleic acid segment substantially as set forth in FIG. 1A, either line 5 or 6, or as disclosed in other areas of the specification herein as coding for a herpes protease or the ICP35 proteins. The nucleotide probe may be labelled. The probe is then incubated with the biological sample to be tested, under selective conditions appropriate for the formation of specific hybrids. The specific hybrids formed between the probes and the nucleic acids of the biological sample are then detected by a variety of methods well known by those skilled in the art, for example, by detecting a radioactive label on the probe. The formation of such hybrids is indicative of the presence of the nucleic acid segment that was sought initially.
A method for treatment of viral infections makes use of the target proteases disclosed herein which are vital to the viral life cycle. An example of such a method comprises preparing an effective mount of an inhibitor of the protease. The amount will depend on the route of treatment. The route may be topical creams, ointments or sprays applied directly to the skin, or intravenous injection for systemic infections. The inhibitor is contemplated to be combined with a pharmacologically acceptable carrier would be appropriate for use in humans depending on the route of application. Finally a therapeutic amount of the inhibitor is determined such that the herpes virus itself is inhibited from reproducing, but the host cells are not destroyed. The method of treatment disclosed herein is particularly applicable to the herpes virus simplex subtypes 1 or 2, but will be generally applicable to the herpes family, members of which are known to have extensive DNA homologies.
This inhibition may be either at the level of transcription, translation, or protein action. Interference with transcription would necessitate interference with mRNA formation on a DNA template. Interference with translation would necessitate interfering with the synthesis of proteins on the mRNA template. Alternatively, the action of the protease may itself be disrupted either by destroying the structure of the protease, in particular its proteolytic domain, altering the cleavage site of it substrate, or by providing a false substrate which inactivates the protease. Another form of inhibitor comprises a nucleic acid segment which is capable of hybridizing with the coding sequence of a herpes protease, but forms a hybrid which inhibits transcription of the mRNA from which the protease would be translated.
There are several inhibitors that appear to be suitable for purposes of this invention. It has been found that chymostatin and diisopropyl fluorophosphate provide 100% inhibition of the protease in an in vitro assay. Phenylmethansulfonyl fluoride provides at least 50% inhibition, this reduction is due to the instability of the inhibitor over time. Other contemplated inhibitors include antipain, aprotinin, leupeptin, (4-amino-phenyl)-methane sulfonyl fluoride, and any other serine protease inhibitors that tests positive in the candidate screening assay described herein. In particular embodiments, non-toxic derivatives of the inhibitors disclosed herein are contemplated.
In order to determine still other inhibitors the candidate substances of interest are screened by preparing a virus protease, combining the protease with the candidate inhibitor substance, and selecting a substrate capable of being cleaved by the protease. The assay is conducted by contacting the substrate with the protease-candidate substance combination, and determining whether the candidate substance has inhibited the action of the protease on the substrate. In an illustrative embodiment, the virus protease used to test for a candidate substance is the purified herpes protease synthesized in vitro in a rabbit reticulocyte lysate by methods disclosed in the present specification.
The protease is combined with the candidate inhibitor substance either in a laboratory in vitro assay or in a test organism. The substrate selected which is capable of being cleaved by the protease, may be the protease itself, or at least the cleavage site of the ICP35 protein precursor c, d. After contacting the substrate with a protease and the candidate inhibitor substance it can be determined whether the substance has inhibited the action of the protease on the substrate by determining whether cleavage of the substrate has taken place. In one embodiment this may be determined by seeing if the ICP35 c, d proteins have produced ICP35 subunits e and f as determined by SDS gel electrophoresis, which are only formed by cleavage of c, d by the protease. If the proteins have not been cleaved, the inference is that the candidate substance indeed inhibits the virus protease. This inhibitor may then be used in therapeutic trials.
The virus protease used in the candidate inhibitor substance assay may be prepared through the application of genetic recombinant technology, wherein, for example, an expression vector includes at least the proteolytic module of the protease. The expression vector is then transferred into an appropriate host cell under conditions which permit expression of the coding sequence. After the sequence has been expressed in the form of a protease or protease segment, the protease may be collected from the cell and further purified if required, by methods well known to those of skill in the art.
An alternative method of preparing the herpes protease is to obtain a sample which contains the protease, for example a herpes infected tissue segment or exudate. The sample is then homogenized and fractionated to obtain a protease fraction. The protease fraction may be then further isolated and purified by methods known to those of skill in the art depending upon the particular application.
This invention also relates to a method for selecting a serine protease with functions equivalent to those disclosed herein in different species of herpes, other non-herpes virus or, indeed, any organism. To select the protease, an amino acid sequence comprising at least the cleavage site of the protease disclosed in the present specification is prepared. The candidate viral protease is then contacted with the cleavage site containing the amino acid sequence which is susceptible to cleavage by a viral serine protease. Finally a determination is made whether cleavage has occurred by using the ICP35 substrate and determining whether ICP35 c, d has been altered to e and f. By these methods, it has been shown that the HSV-2 protease is capable of cleaving ICP35 c, d to e and f.
The methods and compositions of the present invention have made it possible to identify essential serine proteases in other species. The methods of the present invention are generally applicable, only the source genome will change. It is expected that these serine proteases are encoded by conservative segments and are widespread. For the herpes viruses, four of the six viral DNA sequences (HSV-1, EBV, VZV, CMV) are reported and have been entered into a computer data base available to those of skill in the art. Homologies in amino acid sequences and function are expected based on the conservative nature of serine proteases, and homologies detected previously among related species, e.g. the herpes family. (Davison et al., 1986; McGeoch et al., 1988) Thus, it is predictable that UL26 of HSV-1, BVRF2 of EBV, UL80 of CMV, Gene 33 of VZV play the same or similar role in the maturation of capsid and encode a protease. Because extensive sequence homology was found between these presumptive proteases and HSV-1 UL26, it is believed that the action or cleavage mechanism of these protease is the same as the HSV-1 UL26. Thus, it is not surprising that the present invention including inhibitor described have against herpes simplex virus (HSV-1 and HSV-2) and can be applicable to the treatment of other herpes viruses (EBV, VZV, CMV and human herpes virus 6).
As an indication that the serine proteases of the present invention will not be confined to the herpes family, there are reports of capsid assembly in other microbes. Bacteriophage T4 and lambda are most commonly studied for capsid assembly. In these phages, first a preformed capsid is assembled by interaction of outer coat protein and inner scaffolding protein. Then, the scaffolding protein is cleaved by a phage-encoded protease and removed from the capsid. At the same time, the phage DNA is packaged into the capsid to give rise to mature capsid. The cleavage of scaffolding protein is essential to produce mature capsid. Recently, cryo-electromicroscopy studies revealed the similarity of the capsid structure between HSV and lambda phage. ICP35 has been proposed to function as a scaffolding protein in the process of HSV capsid maturation. The cleavage of ICP35 by the proteases of the present invention is required for the capsid maturation and is essential for the replication of the virus. The proteases disclosed herein function as a counterpart to those of phages which cleave the ICP 35 protein to initiate DNA packaging. Therefore, the candidate protease inhibitor assays and methods of treatment disclosed herein have wide applicability than to only the herpes family.
Downstream refers to nucleic acid sequences found in a 3xe2x80x2 direction from a given point of reference along a nucleic acid molecule.
An epitope is an amino acid sequence which is an antigenic determinant.
An open reading frame (ORF) contains a series of triplets coding for amino acids without any termination codons. Sequences of this type are potentially translatable into a protein.
Substantially purified in reference to DNA refers to DNA segments isolated free of their natural state as they may be present in the genome of an organism, and is intended to include segments as they would exist upon genetic engineering, e.g. by insertion into a recombinant vector.
A transcriptional unit is the distance between sites of initiation and termination by RNA polymerase.
Upstream refers to nucleic acid sequences found in a 5xe2x80x2 direction from a given point of reference along a nucleic acid molecule.
A viral protease is an enzyme capable of cleaving viral precursor proteins at a specific site.